U.S. patent application number 14/318112 was filed with the patent office on 2014-10-16 for targeted self-assembly of functionalized carbon nanotubes on tumors.
The applicant listed for this patent is Michael R. McDevitt, J. Justin Mulvey, David A. Scheinberg, Carlos H. Villa. Invention is credited to Michael R. McDevitt, J. Justin Mulvey, David A. Scheinberg, Carlos H. Villa.
Application Number | 20140308203 14/318112 |
Document ID | / |
Family ID | 48698805 |
Filed Date | 2014-10-16 |
United States Patent
Application |
20140308203 |
Kind Code |
A1 |
Scheinberg; David A. ; et
al. |
October 16, 2014 |
Targeted Self-Assembly of Functionalized Carbon Nanotubes on
Tumors
Abstract
Provided herein are methods for delivering a molecule in situ to
a cell and for treating a cancer via the in situ delivery. The
methods comprise contacting or administering to the cell, as two
separate components, a morpholino oligonucleotide comprising a
targeting moiety followed by a single wall nanotube construct
comprising second morpholino oligonucleotides complementary to the
first morpholino oligonucleotides and one or both of a therapeutic
or diagnostic payload molecule linked to the single wall nanotube
construct. Upon self-assembly of a single wall nanotube complex via
hybridization of the first morpholino and second complementary
morpholino oligonucleotides at the cell, the payload molecule is
delivered. Also provided is the two component self-assembly single
wall nanotube system and the single wall nanotube construct
comprising the second component.
Inventors: |
Scheinberg; David A.; (New
York, NY) ; McDevitt; Michael R.; (Bronx, NY)
; Villa; Carlos H.; (Philadelphia, PA) ; Mulvey;
J. Justin; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Scheinberg; David A.
McDevitt; Michael R.
Villa; Carlos H.
Mulvey; J. Justin |
New York
Bronx
Philadelphia
New York |
NY
NY
PA
NY |
US
US
US
US |
|
|
Family ID: |
48698805 |
Appl. No.: |
14/318112 |
Filed: |
June 27, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2012/071915 |
Dec 28, 2012 |
|
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14318112 |
|
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61581228 |
Dec 29, 2011 |
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Current U.S.
Class: |
424/1.49 ;
424/1.73; 424/178.1; 435/375; 514/44A; 530/391.3; 530/391.7;
534/13; 536/24.5; 977/750; 977/842; 977/906 |
Current CPC
Class: |
B82Y 5/00 20130101; C07K
16/2887 20130101; Y10S 977/842 20130101; C12N 2310/113 20130101;
C12N 2320/32 20130101; A61K 51/1248 20130101; A61K 51/1093
20130101; A61K 51/1203 20130101; A61P 1/00 20180101; A61P 43/00
20180101; C12N 2310/351 20130101; A61K 47/6849 20170801; C12N
15/113 20130101; A61P 35/02 20180101; C07K 16/30 20130101; A61P
35/00 20180101; C07K 16/2803 20130101; C12N 15/111 20130101; Y10S
977/75 20130101; Y10S 977/906 20130101; C07K 2317/92 20130101; C12N
2310/3233 20130101; A61K 51/1027 20130101 |
Class at
Publication: |
424/1.49 ;
424/1.73; 530/391.3; 530/391.7; 536/24.5; 534/13; 435/375;
514/44.A; 424/178.1; 977/750; 977/842; 977/906 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 51/10 20060101 A61K051/10; A61K 51/12 20060101
A61K051/12; A61K 47/48 20060101 A61K047/48 |
Goverment Interests
FEDERAL FUNDING LEGEND
[0002] This invention was made with government support under grants
PO1 CA33049, RO1 CA55349, R21CA128406, and GM07739 awarded by the
National Institutes of Health and grant DE-SC0002456 awarded by the
U.S. Department of Energy. The government has certain rights in the
invention.
Claims
1. A method for delivering a molecule in situ to a cell of
interest, comprising: contacting the cell with a first component
comprising a morpholino oligonucleotide linked to a targeting
moiety selective for the cell; contacting the morpholino
oligonucleotide linked to the cell of interest via the targeting
moiety with a second component comprising a soluble single wall
nanotube construct having a plurality of morpholino
oligonucleotides complementary to the targeting moiety-linked
morpholino oligonucleotide and a plurality of the molecules (M*)
independently linked to the single wall nanotube, said molecules
comprising a deliverable payload; and hybridizing the morpholino
oligonucleotide to the complementary morpholino oligonucleotide to
form a self-assembled soluble single wall nanotube complex at the
cell, wherein after hybridization the payload molecules are
delivered to the cell in situ.
2. The method of claim 1, wherein upon self-assembly of the single
wall nanotube complex, antigens located on the targeted cell are
capped and said in situ delivering step comprises internalizing the
complex into the cell.
3. The method of claim 1, wherein both the morpholino
oligonucleotide and the complementary morpholino oligonucleotides
are 18-mer oligonucleotides.
4. The method of claim 3, wherein the morpholino oligonucleotide
has the sequence shown in SEQ ID NO: 1 and the complementary
morpholino oligonucleotide has the sequence shown in SEQ ID NO:
2.
5. The method of claim 1, wherein the targeting moiety is a
monoclonal antibody or fragment thereof or a small molecule ligand,
each selective for a cell associated with a solid or a disseminated
cancer.
6. The method of claim 5, wherein the monoclonal antibody is an
anti-CD20, anti-CD33, or anti-A33 monoclonal antibody or a single
chain variable fragment (scFv) or fragment antigen-binding (Fab)
fragment thereof.
7. The method of claim 5, wherein the monoclonal antibody is the
payload molecule delivered in situ.
8. The method of claim 5, wherein the small molecule ligand is a
folate receptor ligand or an Arg-Gly-Asp peptide.
9. The method of claim 5, where the cancer is a lymphoma, a
leukemia or a colon cancer.
10. The method of claim 1, wherein the payload molecule is one or
both of a diagnostic molecule or a therapeutic molecule.
11. The method of claim 10, wherein the molecule is a radionuclide
linked to the single wall nanotube via a bifunctional chelator.
12. The method of claim 11, wherein the bifunctional chelator is
(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA).
13. The method of claim 10, wherein the diagnostic molecule is
indium-111, copper-64, iodine-124, iodine-131, yttrium-86, a
gadolinium contrast agent, a manganese contrast agent, or a
fluorophore.
14. The method of claim 10, wherein the therapeutic molecule is
actinium-225, astatine-211, technetium-99, lutetium-177,
gallium-68, holmium-166, bismuth-212, bismuth-213, yttrium-90,
copper-67, samarium-117, samarium-153, iodine-123, or
iodine-131.
15. The method of claim 1, wherein the cell of interest is a cell
associated with a cancer in a subject.
16. The method of claim 15, wherein the deliverable payload
comprises molecules diagnostic of the cancer, said method further
comprising detecting the diagnostic payload molecules after in situ
delivery thereof to the cancer-associated cell in the subject,
thereby diagnosing the cancer.
17. The method of claim 15, wherein the deliverable payload
comprises molecules therapeutic against the cancer, said method
further comprising treating the cancer with the therapeutic
molecules after in situ delivery thereof to the cancer-associated
cell in the subject.
18. The method of claim 15, where the cancer is a lymphoma, a
leukemia or a colon cancer.
19. A self-assembly single wall nanotube system, comprising: a
first component having a morpholino oligonucleotide linked to a
monoclonal antibody or fragment thereof; and a second component
having a plurality of complementary morpholino oligonucleotides and
a plurality of one or both of a diagnostic or therapeutic molecules
each independently linked to the single wall nanotube.
20. The self-assembly single wall nanotube system of claim 19,
wherein the diagnostic and therapeutic molecules are a radionuclide
linked to the single wall nanotube via a bifunctional chelator.
21. The self-assembly single wall nanotube system of claim 20,
wherein the bifunctional chelator is
(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA).
22. The self-assembly single wall nanotube system of claim 20,
wherein the radionuclide is actinium-225, astatine-211, indium-111,
technetium-99, lutetium-177, gallium-68, holmium-166, bismuth-212,
bismuth-213, yttrium-86, yttrium-90, copper-64, copper-67,
samarium-117, samarium-153, iodine-123, iodine-124, iodine-125, or
iodine-131.
23. The self-assembly single wall nanotube system of claim 19,
wherein the monoclonal antibody is the therapeutic molecule.
24. The self-assembly single wall nanotube system of claim 19,
wherein the monoclonal antibody is an anti-CD20, anti-CD33, or
anti-A33 antibody or a single chain variable fragment (scFv) or
fragment antigen-binding (Fab) fragment thereof.
25. A single wall nanotube construct, comprising a plurality of a
bifunctional chelator covalently linked to the single wall
nanotube; a radionuclide chelated to each bifunctional chelator;
and a plurality of morpholino oligonucleotides conjugated to the
single wall nanotube.
26. The single wall nanotube construct of claim 25, wherein the
bifunctional chelator is
(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic acid or diethylenetriaminepentaacetic acid (DTPA).
27. The single wall nanotube construct of claim 25, wherein the
radionuclide is actinium-225, astatine-211, indium-111,
technetium-99, lutetium-177, gallium-68, holmium-166, bismuth-212,
bismuth-213, yttrium-86, yttrium-90, copper-64, copper-67,
samarium-117, samarium-153, iodine-123, iodine-124, iodine-125, or
iodine-131.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part under 35 U.S.C.
.sctn.120 of international application PCT/US2012/071915, filed
Dec. 28, 2012, which claims benefit of priority under 35 U.S.C.
.sctn.119(e) to provisional application U.S. Ser. No. 61/581,228,
filed Dec. 29, 2011, now abandoned, the entirety of both of which
are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the fields of
nanomedicine, cancer treatment and tumor targeting. More
specifically, the present invention relates to a self-assembly
single wall nanotube complex functionalized to deliver a molecule
to a cell, for example, a cancer cell, in situ.
[0005] 2. Description of the Related Art
[0006] The rapidly expanding field of nanomedicine has begun to
produce clinical successes in the first generation of engineered
drug nanoparticles (1-2). Nanomedicine involves the use of
synthetic nanoscale particles that aim to take advantage of the
size, shape, and charge of materials to improve drug delivery or
efficacy. In addition, the intrinsic properties of some
nanomaterials result in unique physicochemical properties. Carbon
nanotubes have seen widening application in biomedical research
(3), in part because of the potential to append a diverse set of
ligands, including small molecules (4), peptides (5-6),
oligonucleotides (7), radioisotopes (8), monoclonal antibodies
(9-10), and other targeting moieties.
[0007] The pharmacokinetics and pharmacodynamics of carbon
nanotubes appear to depend highly on the chemical approaches used
to render them water dispersable and biocompatible (11-12).
Functionalization and dispersion imparts stability in aqueous
environments and mitigates potential toxic effects (13). An
important feature of covalently functionalized single-walled carbon
nanotubes that allows their use as drug carriers is their rapid
renal clearance via longitudinal renal glomerular filtration
despite apparently large molecular weights (14-16). This clearance
phenomenon has been termed fibrillar pharmacology and directly
contrasts with the pharmacokinetic profiles of globular
proteins.
[0008] Typically, in systemic targeting of malignancies, drug
delivery vehicles such as mAb or nanoparticles are appended with
cytotoxic effectors such as a chemotherapeutic small molecule or
radioisotopes in a "single-step" process. However, prolonged
half-lives of the effector molecules in vivo, especially those
unbound or in excess, will increase toxicity. In contrast, a short
circulation time of the agent is problematic as it reduces the time
in which a high enough concentration can be maintained in the
bloodstream to drive tumor penetration and cell binding.
[0009] Two step targeting or pre-targeting separates the required,
slow, non-toxic tumor-targeting process of the vehicle from the
necessary rapid clearance of the cytotoxic agent to achieve the
desired pharmacokinetic goals (17). In this approach, a
long-circulating tumor selective agent, such as a monoclonal
antibody, is first administered and allowed a sufficient
circulation time, typically 2-5 days, to accumulate at the tumor
and clear the bloodstream. This is followed by a rapidly cleared,
i.e., half life of <10 min, second-step agent, armed with the
cytotoxic or diagnostic effector, that has a high-affinity
interaction with the initial agent. This interaction may involve
streptavidin-biotin (18), bispecific antibody-hapten (19),
oligonculeotide hybridization (20), or, more recently, covalent
`click` chemistry (21). The second agent rapidly equilibrates with
the initial agent, while also clearing the bloodstream. This
approach allows for improved ratios of the cytotoxic or imaging
agent in the tumor to that in the blood and the corresponding `area
under the curve` in concentration versus time, and in other
off-site tissues. Pretargeting approaches have been previously
explored (22-24), but did not include a second step reagent that
both delivered a large payload and cleared quickly, enhancing the
therapeutic index.
[0010] Because the second step in such a strategy requires rapid
clearance, this agent is nearly always a small molecule such as a
modified biotin, or a lone, short oligonucleotide. However, small
molecules limit potency and sensitivity, as they are usually
mono-substituted with the therapeutic or imaging agent,
respectively. A highly multivalent effector as a second step should
allow for amplification of signal, such as a toxin or diagnostic
nuclide, multi-functionality, as well as improved binding affinity
(25-26). However, such multivalent constructs as a second step are
often too large to allow for the rapid clearance time necessary to
avoid toxicity. Therefore, a covalently modified single-walled
carbon nanotubes may offer the unique advantage of being highly
multivalent and multi-functional while maintaining their rapid
clearance.
[0011] Thus, there is a recognized need in the art for improved
methods for multi-step delivery of a diagnostic and/or a
therapeutic molecule or compound using single wall nanotube
constructs. The prior art is deficient in the lack of self-assembly
single wall nanotube systems effective to deliver a diagnostic
and/or therapeutic compound via a two step-cell targeting method
without prolonged exposure to a cytotoxic agent during targeting of
the cell. The present invention fulfills this longstanding need and
desire in the art.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to a method of delivering
a molecule in situ to a cell of interest. The method comprises
contacting the cell with a first component comprising a morpholino
oligonucleotide linked to a targeting moiety selective for the cell
and contacting the morpholino oligonucleotide linked to the cell of
interest via the targeting moiety with a second component
comprising a soluble single wall nanotube construct having a
plurality of morpholino oligonucleotides complementary to the
targeting moiety-linked morpholino oligonucleotide and a plurality
of the molecules independently linked to the SWNT such that the
molecules comprise a deliverable payload. The morpholino
oligonucleotide hybridizes to the complementary morpholino
oligonucleotide to form a self-assembled soluble soluble single
wall nanotube complex at the cell, wherein after hybridization the
payload molecules are delivered in situ to the cell.
[0013] The present invention also is directed to a related method
for diagnosing a cancer in a subject. In the method the cell of
interest is a cell associated with cancer in a subject and the
deliverable payload comprises molecules diagnostic of the cancer.
The method further comprises comprising detecting the diagnostic
payload molecules after in situ delivery thereof to the
cancer-associated cell in the subject, thereby diagnosing the
cancer.
[0014] The present invention also is directed to another related
method for treating a cancer in a subject. In the method the cell
of interest is a cell associated with cancer in a subject and the
deliverable payload comprises molecules therapeutic against the
cancer. The method further comprises treating the cancer with the
therapeutic molecules after in situ delivery thereof to the
cancer-associated cell in the subject.
[0015] The present invention is directed further to a self-assembly
single walled nanotube (SWNT) system. The self-assembly SWNT system
is a two-component system. The first component has a morpholino
oligonucleotide linked to a monoclonal antibody. The second
component has a plurality of complementary morpholino
oligonucleotides and a plurality of one or both of a diagnostic or
therapeutic molecules each independently linked to the SWNT.
[0016] The present invention is directed further still to a single
wall nanotube construct. The single wall nanotube construct
comprises a plurality of a bifunctional chelator covalently linked
to the single wall nanotube, a radionuclide chelated to each
bifunctional chelator and a plurality of morpholino
oligonucleotides conjugated to the single wall nanotube.
[0017] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention. These
embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions and certain embodiments of the
invention briefly summarized above are illustrated in the appended
drawings. These drawings form a part of the specification. It is to
be noted, however, that the appended drawings illustrate preferred
embodiments of the invention and therefore are not to be considered
limiting in their scope.
[0019] FIGS. 1A-1D depict the design and HPLC characterization of
self-assembling SWNT-cMORF constructs. FIGS. 1A-1B show a synthetic
scheme for chemical functionalization of SWNT-NH.sub.2 to produce
radio- and fluorescent labeled SWNT-cMORF conjugates. FIG. 1C is a
3D gel permeation HPLC chromatograph of compound 3
(SWNT-cMORF-NH.sub.2) tracing the spectrum of eluted material
across time. Only a single peak is evident and the spectrum is
consistent with single-walled carbon nanotubes modified with
bis-aryl hydrazone groups (shoulder at .lamda.=.about.354 nm) and
decreasing absorption through 600 nm, which is a quality of all
nanotubes. FIG. 1D is a diagrammatic representation (not to scale)
of SWNT-cMORF self-assembly onto mAb-MORF targeted tumor cells. The
triangle is tumor antigen; the dot is appended cytotoxic or
diagnostic moiety.
[0020] FIG. 2A-2E depict that SWNT-cMORF hybridizes with multiple
mAb-MORF in vitro and in the presence of serum. FIG. 2A shows a
size exclusion HPLC of SWNT-cMORF alone (right peak),
anti-CD20-MORF alone (middle peak), and SWNT-cMORF mixed with
anti-CD20-MORF (left peak). FIG. 2B that a similar result is
obtained when combining the SWNT-cMORF-.sup.111In(DOTA) with
anti-CD20-MORF and tracking the radiolabel associated with the
either the SWNT-cMORF alone (solid line) or when mixed with the
antibody (dashed line). FIG. 2C shows similar peak shift patterns
are seen with SWNT-cMORF-.sup.111In(DOTA) mixed with anti-A33-MORF
antibodies, demonstrating that the complex formation is independent
of antibody identity. FIG. 2D shows that the complex formation
still occurred when the two components were mixed in the presence
of 100% serum (dashed line) and this could be blocked when excess
free cMORF was added to the mixture (solid line). FIG. 2E shows
that SWNT-cMORF-.sup.111In(DOTA) could be captured by protein A
beads only when a complementary mAb-MORF was added.
[0021] FIGS. 3A-3E depict the self-assembly of SWNT-cMORF onto
tumor cells pretargeted with mAb-MORF is highly specific and high
affinity. FIG. 3A shows quantitation of flow cytometric assay of
binding of SWNT-cMORF-AF647 onto HL60 cells. Pretreatment was with
HL60 specific anti-CD33-MORF, isotype control anti-CD20-MORF, or
specific anti-CD33-MORF+blocking with excess free cMORF. Data is
presented as the change in median fluorescence. FIG. 3B shows
quantitation of flow cytometric assay of binding of
SWNT-cMORF-AF647 onto DAUDI cells. Pretreatment was with DAUDI
specific anti-CD20-MORF, isotype control anti-CD33-MORF, or
specific anti-CD20-MORF+blocking with excess free cMORF. FIG. 3C
shows quantitation of flow cytometric assay of binding of
SWNT-cMORF-AF647 onto LS174T cells. Pretreatment was with LS174T
specific anti-A33-MORF, isotype control anti-CD33-MORF, or specific
anti-A33-MORF+blocking with excess free cMORF. FIG. 3D shows a
representative flow cytometric histogram of cell binding assay with
SWNT-cMORF-AF647 on untreated (red), isotype control pretargeted
(black), specific pretargeted+cMORF block (blue), or specific
pretargeted (green) cells. FIG. 3E shows a binding curve for
SWNT-cMORF-AF647 onto anti-CD20 pretargeted Daudi cells (squares)
and anti-A33 pretargeted LS174T cells (triangles). Curves were fit
using GraphPad Prism using an algorithm for one-site specific
binding with variable Hill slope (R.sup.2=0.95, 0.97).
[0022] FIGS. 4A-4E demonstrate that SWNT-cMORF conjugates are able
to induce antigen capping and internalization when self-assembled
onto cells targeted with mAb-MORF. FIG. 4A shows that anti-A33 (row
2) remained stably bound to LS174T cells (row 1, DAPI nuclear
stain) at 37 8 C for up to 24 h (4 h is shown) and were evenly
distributed about the cell membrane. FIG. 4B shows that
anti-A33-MORF conjugates (rows 2 and 5) exhibited similar surface
stability when bound to LS174T cells (blue, rows 1 and 3).
High-power images are provided (rows 4, 5 and 6), as well 647 nm
views showing the absence signal (rows 3 and 6). FIG. 4C shows that
cells pretreated with A33-MORF (rows 2 and 5) followed by
SWNT-cMORF-AF647 (rows 3 and 6), allowing for self-assembly at 37 8
C for up to 4 h, demonstrated a significant change in the
distribution of the bound antibody with a change to scattered
punctate staining. A similar pattern was observed for 30 min and 1
h incubations. High-power images are provided (rows 4,5 and 6).
FIG. 4D shows that cells treated with anti-A33-MORF (rows 2 and 5)
followed by cMORF-AF647 (rows 3 and 6) alone did not demonstrate a
change in distribution of A33 about the cell membrane. High-power
images are provided (rows 4,5 and 6). Anti-A33-MORF was evenly
distributed about the plasma membrane, and cMORF-AF647 co-localized
with the targeted mAb-MORF. FIG. 4E shows composite staining of
conditions in rows 2 and 3 in FIG. 4C.
[0023] FIGS. 5A-5D depict that SWNT-cMORF can selectively
self-assemble onto pretargeted tumors in vivo. FIG. 5A is a flow
cytometric analysis of CD20-positive, CD33-negative Daudi-GFP cells
collected from mice treated with either isotype control
anti-CD33-MORF or tumour-specific anti-CD20-MORF. Data show the
shift in median fluorescence from untreated Daudi-GFP cells
(representative mice are shown). FIG. 5B is a representative flow
cytometric histograms of data in FIG. 5A showing untreated cells
(front peak), isotype mAb-MORF treated cells (middle peak), and
specific mAb-MORF treated cells (back peak). FIG. 5C shows tumor to
blood ratios (T:B) for LS174T tumored mice pretargeted with either
specific anti-A33-MORF or isotype control anti-CD33-MORF followed
by injection of SWNT-cMORF-.sup.111In(DOTA). Signal measurements in
% ID per gram were measured at 4 or 24 h post-injection of
SWNT-cMORF-.sup.111In(DOTA). There was a significant increase
(p<0.05) in tumor accumulation of the
SWNT-cMORF-.sup.111In(DOTA) at the tumor site only pre-targeted
with specific antibody compared to isotype control. FIG. 5D shows
the ratio of activity.+-.SD of tumor-specific and isotype
pre-targeted antibodies followed by second step
SWNT-cMORF-.sup.111In(DOTA) treatment at 24 hours on off-target
tissues (n=4) 24 hours post treatment.
[0024] FIGS. 6A-6C shows that SWNT-cMORF-.sup.225Ac(DOTA) mitigates
radioisotope toxicity and can be used as an effective agent in
multistep therapy of disseminated lymphoma. FIG. 6A shows whole
animal weights of tumor-free mice treated with varying dose levels
of .sup.225Ac labeled SWNT-cMORF-DOTA. An additional group of mice
received 450 nCi of free .sup.225Ac as a control. Data reflect
average weights, n=5 for all groups. Animal deaths are noted by an
asterisk. FIG. 6B is a Kaplan-Meier plot of pre-annealed
single-step treatment mouse survival with comparison to not
annealed 1,800 nCi group from a. The toxicity study was halted at
40 days. Median survival was less than 2 weeks in all groups. FIG.
6C shows averaged organ weights of surviving mice sacrificed at 140
days post exposure to SWCNT-cMORF-.sup.225Ac(DOTA) organized by
treatment group.
[0025] FIGS. 7A-7B illustrate the efficacy of
SWNT-cMORF-(.sup.225Ac)DOTA (FIG. 7A) compared to controls (FIG.
7B) in multi-step therapy of disseminated lymphoma. Mice previously
implanted with luciferase-transfected Daudi lymphoma cells that
were injected with 5 mg/ml luciferin and were imaged after a 5
minute delay. Luminescence scale is the same for all images. The
parameters are equivalent for all images, but, as a tradeoff, this
leads to saturation that disallows quantification. Mice were
treated with multistep therapy as previously noted and imaged at
days 0, 3, 6, and 15 after treatment. Growth, rituximab therapy,
isotype high-dose radiation and blocked two-step controls are
provided.
DETAILED DESCRIPTION OF THE INVENTION
[0026] As used herein, the term, "a" or "an" may mean one or more.
As used herein in the claim(s), when used in conjunction with the
word "comprising", the words "a" or "an" may mean one or more than
one. As used herein "another" or "other" may mean at least a second
or more of the same or different claim element or components
thereof.
[0027] As used herein, the term "or" in the claims refers to
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or".
[0028] As used herein "another" or "other" may mean at least a
second or more of the same or different claim element or components
thereof. "Comprise" means "include."
[0029] As used herein, the term "about" refers to a numeric value,
including, for example, whole numbers, fractions, and percentages,
whether or not explicitly indicated. The term "about" generally
refers to a range of numerical values (e.g., +/-5-10% of the
recited value) that one of ordinary skill in the art would consider
equivalent to the recited value (e.g., having the same function or
result). In some instances, the term "about" may include numerical
values that are rounded to the nearest significant figure.
[0030] As used herein, the term "contacting" refers to any suitable
method of bringing one or both of the two components comprising a
self-assemblying single wall nanotube system, as described herein,
into contact with a cell or an antigen comprising the same. In
vitro or ex vivo this is achieved by exposing the cell to the
components in a suitable medium. For in vivo applications, any
known method of administration is suitable.
[0031] As used herein, the term "SWNT" refers to a single wall
nanotube (SWNT) that has a functional pendant moiety or handle,
such as a sidewall amine or aldehyde carbonyl, suitable for
bioconjugation.
[0032] As used herein, the terms "MORF" and "cMORF" refer to a
synthetic DNA analog with a morpholino backbone or morpholino
oligonucleotide. The sequence of a cMORF oligonucleotide is
complementary to that of a MORF oligonucleotide. As such, the terms
"mAB-MORF" or "MORF-mAB" refers to a morpholino oligonucleotide
linked to a monoclonal antibody via standard chemical methods.
[0033] As used herein, "M*" refers to a chelatable radiometal or
other radionuclide or a chelatable contrast agent such as a
nonradioactive nuclide useful as therapeutics for or diagnostics of
a pathophysiological condition, for example, a cancer. As such,
"M*-DOTA" refers to a radiometal chelated to the bifunctional
chelator
(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA) which
itself is directly linked to the SWNT.
[0034] As used herein, the term "SWNT-cMORF" refers to a single
wall nanotube having a plurality of morpholino oligonucleotides
with sequences complementary to the MORF oligonucleotides
comprising a mAB-MORF oligonucleotide. As such, the term
"SWNT-(cMORF-(mAB-MORF))" refers to a self-assembled complex in
which the cMORF oligonucleotide is hybridized to the MORF
oligonucleotide.
[0035] As used herein, SWNT-(M*DOTA)-(cMORF-(mAB-MORF))" refers to
the self-assembled SWNT-(cMORF-(mAB-MORF)) complex in which the
single wall nanotube also comprises a radiometal or other
radionuclide or chelatable contrast agent linked to the SWNT via
the bifunctional chelator
(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA).
[0036] As used herein, the term "subject" refers to any recipient
of the SWNT self-assemblying components or SWNT constructs as
described herein.
[0037] In one embodiment of the present invention there is provided
a method of delivering a molecule in situ to a cell of interest,
comprising contacting the cell with a first component comprising a
morpholino oligonucleotide linked to a targeting moiety selective
for the cell; contacting the morpholino oligonucleotide linked to
the cell of interest via the targeting moiety with a second
component comprising a soluble single wall nanotube construct
having a plurality of morpholino oligonucleotides complementary to
the targeting moiety-linked morpholino oligonucleotide and a
plurality of the molecules (M*) independently linked to the single
wall nanotube, said molecules comprising a deliverable payload; and
hybridizing the morpholino oligonucleotide to the complementary
morpholino oligonucleotide to form a self-assembled soluble single
wall nanotube complex at the cell; wherein after hybridization the
payload molecules are delivered in situ to the cell. In an aspect
of this embodiment the in situ delivering step may comprise capping
antigens located on the targeted cell via the self-assembled
SWNT-mAB complex; and internalizing the complex into the cell.
[0038] In this embodiment upon self-assembly of the single wall
nanotube complex, antigens located on the targeted cell may be
capped and the in situ delivering step may comprise internalizing
the complex into the cell. Also, both the morpholino
oligonucleotide and the complementary morpholino oligonucleotides
may be 18-mer oligonucleotides. Particularly, the morpholino
oligonucleotide may have the sequence shown in SEQ ID NO: 1 and the
complementary morpholino oligonucleotide may have the sequence
shown in SEQ ID NO: 2.
[0039] Also in this embodiment, the targeting moiety may be a
monoclonal antibody or fragment thereof or a small molecule ligand
each selective for a cell associated with a solid or a disseminated
cancer. In one aspect the monoclonal antibody may be an anti-CD20,
anti-CD33, or anti-A33 monoclonal antibody or a single chain
variable fragment (scFv) or fragment antigen-binding (Fab) fragment
thereof. In another aspect the monoclonal antibody itself may be
the payload molecule delivered in situ. In yet another aspect the
small molecule ligand may be a folate receptor ligand or an
Arg-Gly-Asp peptide. Representative examples of a cancer are a
lymphoma, a leukemia or a colon cancer.
[0040] In addition, the payload molecule may be one or both of a
diagnostic molecule or a therapeutic molecule. The molecule may be
a radionuclide linked to the SWNT via a bifunctional chelator.
Representative examples of a bifunctional chelator are
(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA). In
one aspect the diagnostic molecule may be indium-111, copper-64,
iodine-124, iodine-131, yttrium-86, a gadolinium contrast agent, a
manganese contrast agent, or a fluorophore. In another aspect the
therapeutic molecule may be actinium-225, astatine-211,
technetium-99, lutetium-177, gallium-68, holmium-166, bismuth-212,
bismuth-213, yttrium-90, copper-67, samarium-117, samarium-153,
iodine-123, iodine-125, or iodine-131.
[0041] In another embodiment of the present invention there is
provided a method for diagnosing a cancer in a subject, comprising
administering sequentially to the subject a first component of a
self-assembled single wall nanotube complex having a targeting
moiety, selective for a cell associated with the cancer, conjugated
to a morpholino oligonucleotide and a second component thereof
comprising a single wall nanotube construct having a plurality of
complementary morpholino oligonucleotides and of diagnostic payload
molecules independently conjugated thereto; and detecting the
diagnostic payload molecules in the subject after self-assembly of
the first and second components of the complex at the
cancer-associated cell, thereby diagnosing the cancer.
[0042] In this embodiment the first component morpholino
oligonucleotides may have a sequence shown in SEQ ID NO: 1 and the
second component complementary morpholino oligonucleotides may have
a sequence shown in SEQ ID NO: 2. Also, the targeting moiety may be
a monoclonal antibody or fragment thereof or a small molecule
ligand. Representative examples of the monoclonal antibody or
fragment thereof and the small molecule ligand are as described
supra. In addition the diagnostic payload may be linked to the
single wall nanotube via a bifunctional chelator. In addition, the
bifunctional chelators and the cancers are as described supra.
Furthermore, the diagnostic payload molecule may be indium-111,
copper-64, iodine-124, iodine-131, yttrium-86, a gadolinium
contrast agent, a manganese contrast agent, or a fluorophore.
[0043] In this embodiment the cell of interest may be a cell
associated with a cancer in a subject. In one aspect the
deliverable payload may comprise molecules diagnostic of the
cancer, where the method further comprises detecting the diagnostic
payload molecules after in situ delivery thereof to the
cancer-associated cell in the subject, thereby diagnosing the
cancer. In another aspect, the deliverable payload may comprise
molecules therapeutic against the cancer, where the method further
comprises treating the cancer with the therapeutic molecules after
in situ delivery thereof to the cancer-associated cell in the
subject. In all embodiments and aspects thereof, the cancer may be
a lymphoma, a leukemia or a colon cancer.
[0044] In yet another embodiment of the present invention there is
provided a method for treating a cancer in a subject, comprising
administering sequentially to the subject a first component and a
second component of a self-assembled single wall nanotube complex
functionalized with a targeting moiety and one or more therapeutic
payload molecules independently linked to morpholino
oligonucleotides; where the first and second components
self-assemble on a cell associated with the cancer that is targeted
by the targeting moiety, thereby delivering the therapeutic payload
to the cell to treat the cancer.
[0045] In one aspect the first component of the self-assembled SWNT
complex may comprise a first morpholino oligonucleotide conjugated
to the targeting moiety. The first morpholino oligonucleotide may
have a sequence shown in SEQ ID NO: 1. In another aspect the second
component of the self-assembled single wall nanotube complex may
comprise a plurality of second morpholino oligonucleotides having a
sequence complementary to the first morpholino oligonucleotide and
a plurality of the payload molecules independently linked to the
soluble single wall nanotube. The second complementary morpholino
oligonucleotide may have a sequence shown in SEQ ID NO: 2.
[0046] In all embodiments and aspects thereof upon self-assembly of
the complex, antigens located on the cancer-associated cell may be
capped and the complex may be internalized into the cell. Also, the
therapeutic payload may be a radionuclide linked to the single wall
nanotube via a bifunctional chelator. A representative example of a
bifunctional chelator is as described supra. The therapeutic
radionuclide may be actinium-225, astatine-211, technetium-99,
lutetium-177, gallium-68, holmium-166, bismuth-212, bismuth-213,
yttrium-90, copper-64, copper-67, samarium-117, samarium-153,
iodine-123, iodine-125, or iodine-131. Alternatively, the
monoclonal antibody may be the therapeutic payload molecule. The
targeting moiety, including monoclonal antibodies or fragments
thereof and small molecule ligands and the cancers are as described
supra
[0047] In yet another embodiment of the present invention there is
provided a self-assembly single walled nanotube complex, comprising
a first component having a morpholino oligonucleotide linked to a
monoclonal antibody or a fragment thereof; and a second component
having a plurality of complementary morpholino oligonucleotides and
a plurality of one or both of a diagnostic or therapeutic molecules
each independently linked to the single wall nanotube.
[0048] In this embodiment the diagnostic and therapeutic molecules
may be a radionuclide linked to the single wall nanotube via a
bifunctional chelator, such as DOTA or DTPA, as described supra.
Representative examples of the radionuclide are actinium-225,
astatine-211, indium-111, technetium-99, lutetium-177, gallium-68,
holmium-166, bismuth-212, bismuth-213, yttrium-86, yttrium-90,
copper-64, copper-67, samarium-117, samarium-153, iodine-123,
iodine-124, iodine-125, or iodine-131. In addition the monoclonal
antibody may be the therapeutic molecule. Examples of a monoclonal
antibody or fragment thereof may be an anti-CD20, anti-CD33, or
anti-A33 antibody or a single chain variable fragment (scFv) or
fragment antigen-binding (Fab) fragment thereof.
[0049] In yet another embodiment of the present invention there is
provided a single wall nanotube construct comprising a plurality of
a bifunctional chelator covalently linked to the single wall
nanotube; a radionuclide chelated to each bifunctional chelator;
and a plurality of morpholino oligonucleotides conjugated to the
single wall nanotube. In this embodiment the bifunctional chelator
and the radionuclide for a diagnostic or a therapetic molecule or a
payload molecule are as described supra.
[0050] Provided herein are single wall nanotube constructs, systems
and methods useful in multistep self-assembly approaches for
delivering molecules or payloads to a cell. The conjugation of
single-walled carbon nanotubes to morpholino oligonucleotide
sequences (SWNT-cMORF) confers on them the ability to self-assemble
with cancer-selective antibodies appended with complementary
oligonucleotides (mAb-MORF) with sub-nanomolar affinity and in
physiologic conditions. Self-assembly promotes target antigen
complex internalization that enables delivery of diagnostic and/or
therapeutic agents into cells via normally non-internalizing
targets.
[0051] Generally, the self-assembly single wall nanotube (SWNT)
system comprises two components. The first component comprises a
morpholino oligonucleotide linked to a targeting moiety, such as a
monoclonal antibody or fragment thereof or other targeting peptide
or ligand. Preferably, the targeting moiety is a monoclonal
antibody or a single chain variable fragment (scFv) or fragment
antigen-binding (Fab) fragment thereof and the first component has
the structure mAb-MORF or MORF-mAb. Alternatively, the targeting
moiety may be a small molecule ligand, such as, but not limited to,
an Arg-Gly-Asp (RGD) peptide or a folate receptor ligand. The
targeting moiety may be linked to the morpholino oligonucleotide
via standard chemical methods. Preferably, the targeting moiety
will target a cell associated with a cancer.
[0052] The second component in the system comprises synthetic,
covalently modified SWNTs bearing multiple copies of a synthetic
oligonucleotide analog, for example, morpholino oligonucleotides
(cMORF) and fluorophores and/or radioisotopes or other nuclides.
The linkage between the SWNT and morpholinos (SWNT-cMORF) is
spectroscopically quantifiable with about 5-15 morpholinos per 200
nm of SWNT. The SWNT-cMORF conjugates can self-assemble onto
antibody targeted cancer cell surfaces with excellent specificity
and high affinity via hybridization to the morpholino
oligonucleotide MORF.
[0053] The morpholino oligonucleotide and its complement may be
short oligonucleotides, such as an 18-mer oligonucleotide, for
example, but not limited to, the complementary sequences in SEQ ID
NOS: 1 and 2, as shown in Example 1. Morpholino oligonucleotides
are less immunogenic (28) than protein based pairs. This allows for
multiple administrations and potentially more complex multi-step
self-assembling constructs, complexes and systems.
[0054] The multivalent self-assembly system can trigger antigen
capping and internalization of mAb-MORF targets, even in otherwise
surface stable antigens. This internalization may be a result of
the capability of SWNT-cMORF to self-assemble with multiple
mAb-MORF targets, mimicking cross-linking of surface receptors. The
ability to trigger internalization of surface antigens through
self-assembly of the system components may enhance therapeutic
efficacy of agents appended to the SWNT. Thus, the second step in
the self-assembly also may trigger internalization of the initial
targeting agent thereby capturing the cytotoxic agent within the
target cell and improving the therapeutic index.
[0055] As such, the self-assembly constructs, systems and complexes
of the present invention are effective vehicles in methods for
treating a pathophysiological state, such as a solid or a
disseminated cancer. Representative solid and disseminated cancers
may be, but not limited to, colon cancers, lymphomas and leukemias.
Therapeutic radionuclides may be delivered to cancer cells using
the SWNT-cMORF constructs. The therapeutic radionuclide may be, but
not limited to, actinium-225, astatine-211, technetium-99,
lutetium-177, gallium-68, holmium-166, bismuth-212, bismuth-213,
yttrium-90, copper-67, samarium-117, samarium-153, iodine-123,
iodine-125, or iodine-131.
[0056] Also, the self-assembly constructs, systems and complexes
are useful for detecting and diagnosing a pathophysiological state
or condition in vivo or in vitro in diagnostic assays. The
SWNT-cMORF may comprise a contrast agent, a diagnostic radionuclide
or a fluorophore linked to the SWNT. For example a diagnostic
radionuclide may be, but not limited to, indium-111, copper-64,
iodine-124, iodine-131, or yttrium-86. Other non-radionuclide
diagnostic agents may be a gadolinium contrast agent, a manganese
contrast agent or a fluorophore, as are known in the art. It is
contemplated that superparamagnetic iron oxide based contrast
agents also may be utilized as diagnostic agents in the methods and
compositions provided herein.
[0057] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
Example 1
Methods and Materials
Modification of SWNT
[0058] High purity (>90%) single walled carbon nanotubes were
obtained from NanoLab Inc (Waltham, Mass.). These arc-discharge
produced single-walled carbon nanotubes were reacted according to
described protocols (33) to produce SWNT-NH.sub.2. The
SWNT-NH.sub.2 product was purified on a C18 Seppak (Waters) by
application and wash in 20% acetonitrile: 0.1 M triethyl amine
acetate. The purified product was eluted with 50%
acetonitrile:water. This solution was then lyophilized to give the
dark brown SWNT-NH2 solid, or diluted directly into a bicarbonate
buffer (0.1 M Na HCO3, pH 9). To the SWNT-NH2 was added 0.6 mmol/g
of PEG4/PFB (Solulink) long chain crosslinker. This reaction was
allowed to proceed for 2 hours at room temperature. The reaction
mixture was then purified on a disposable benchtop 10-DG size
exclusion column (Biorad), with elution of the aldehyde and amine
bearing SWNT-4FB-NH.sub.2 (2) product into a 0.1 M MES, 0.15 M
NaCl, pH 5.5 buffer.
Conjugation of MORF and cMORF to Nanotubes and Antibodies
[0059] Morpholino oligonucleotides MORF (TCTTCTACTTCACAACTA, SEQ ID
NO: 1) and cMORF (TAGTTGTGAAGTAGAAGA, SEQ ID NO: 2) were custom
synthesized (Gene Tools Inc.), and contained primary amines on the
3' end. The primary amine was capped with either an aldehyde or
hydrazine moiety for conjugation to the antibodies or nanotubes,
respectively. The MORF-NH.sub.2/cMORF-NH.sub.2 were reacted with a
20-fold excess of succinimidyl 4-formyl benzoate (Thermo
Scientific) or succinimidyl hydrazinonicotinamide (Thermo
Scientific) in a 0.1 M sodium bicarbonate buffer containing 20%
acetonitrile. Free linker was removed through gel-filtration
chromatography on a 10-DG column (Bio-Rad) and the morpholinos were
purified into a buffer of 0.1 M MES, 0.15 M NaCl, pH 5.5.
[0060] The cMORF-HyNic was mixed with SWNT-4FB-NH.sub.2 at a ratio
of 0.2 mmol of cMORF per gram of single-walled carbon nanotubes in
a buffer of 0.1 M MES, 0.15 M NaCl, pH 5.5 containing 25%
acetonitrile. The reaction was allowed to proceed at 37.degree. C.
for 6 hours, and then at room temperature overnight with shaking.
The SWNT-cMORF-NH.sub.2 product was purified by washing on a C18
Seppak (Waters) with 25% acetonitrile in 0.1 M TEAA. The product
was eluted with 50% acetonitrile:water and then lyophilized to give
the solid product. The product was verified to be free of
cMORF-HyNic by reverse phase C18 HPLC.
[0061] Monoclonal antibodies HuM195 (Lintuzumab;
Sloan-Kettering)/anti-CD33, Rituximab/anti-CD20 (Genentech), and
huA33/anti-A33 (Ludwig Institute) were diluted into a modification
buffer of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.6. A 10 to 15
fold excess of succinimidyl hydrazine nicotinamide was added slowly
to the antibody solutions. The antibodies were reacted for 2 hours
followed by purification on a 10-DG gel filtration column. The
hydrazone modified mAbs (mAb-HyNic) were eluted into 0.1 M MES,
0.15 M NaCl, pH 5.5. The mAb-HyNic were then reacted with the
aldehyde modified MORF-4FB at a ratio of 8 MORF per antibody and
reacted at room temperature for at least 12 hours with gentle
shaking. The remaining free MORF-4FB was removed by purification on
a 100,000 MWCO centrifugal filter device (Amicon Ultra, Millipore),
with at least three washes in PBS. Protein was quantified through
the D.sub.C protein assay (Biorad) and attached morpholinos were
quantified spectroscopically by the bis-aryl hydrazone chromophore
(.lamda..sub.max=354 nm). The purity of the mAb-MORF conjugates
were verified by HPLC on a Superdex 200 column.
Radiolabeling
[0062] The SWNT-cMORF-NH.sub.2 was dissolved in a buffer of 0.1 M
sodium bicarbonate previously treated with Chelex 100 resin
(Bio-Rad) to render the buffer free of divalent metals. To this
solution was added a ratio of 10 mmol/g of amine reactive
2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraa-
cetic acid (DOTA-SCN, Macrocyclics). This reaction was kept at room
temperature for 2 hours followed by purification on a 10-DG
gel-filtration column previously treated with 25 mM DTPA to render
the stationary phase metal free. The product was eluted in
metal-free distilled H.sub.2O and lyophilized to give the solid
SWNT-cMORF-DOTA product. This product was radiolabeled with
.sup.111InCl.sub.3 (MDS Nordion) by addition of radioisotope in an
ammonium acetate buffer (1M), pH 5, containing 20% acetonitrile for
a period of 0.5 hours at 50.degree. C. The labeling mixture was
quenched by addition of 50 mM DTPA and purified on a 10-DG gel
filtration column into PBS. The radiochemical purity was measured
by instant thin layer chromatography using mobile phases of 10 mM
EDTA and 0.9% NaCL/10 mM NaOH (8). ITLC strips were counted using a
System 400 Imaging Scanner (Bioscan, Inc.) Radiochemical purity was
>90% across 5 repeated labeling reactions and was confirmed by
radio-detection on HPLC. The cMORF-DOTA construct used in
comparative binding assays was similarly labeled with .sup.111In
(0.8 Ci/g) and alternatively with .sup.225Ac (0.66 Ci/g).
Radiochemical purity was quantitative.
HPLC Assays
[0063] All high performance liquid chromatography was performed on
a System Gold Bioessential 125/168 diode-array detection instrument
(Beckman Coulter) equipped with an in-line gamma-RAM model 3
radioactivity detector (IN/US). Analysis was performed using both
32 Karat chromatography software (Beckman Coulter) and Prism
graphing and analysis software (Graphpad). Gel filtration
chromatography was performed on a Superdex 200 column in a 20 mM
sodium acetate, 0.15 M sodium chloride, pH 6.5 isocratic mobile
phase. Reverse phase HPLC was performed on a Gemini C18 column
(Phenomenex) with a gradient of 20% acetonitrile in 0.1 M
tetraethyl ammonium acetate (TEAA) to 100% acetonitrile. For
antibody-nanotube hybridization assays, samples were mixed in
phosphate buffered saline, unless otherwise noted, in 150 .mu.L
total volume for 5 minutes prior to injection onto the HPLC
column.
Confocal Microscopy
[0064] LS174T cells were seeded onto tissue culture treated,
poly-lysine coated, multi-well chambered slides (Nunc) for 24 hours
at 50,000 cell/mL. The slides were treated with 10 nM of one of
anti-A33-MORF, anti-A33, or control anti-CD19-MORF antibodies for 4
hours at 37.degree. C. The unbound antibody was then washed out,
and cells were treated with media alone, media containing 7.5
.mu.g/mL SWNT-cMORF-AF647 conjugates, or media containing 100 nM
fluorescently labeled cMORF-AF647. The cells were then incubated at
37.degree. C. for up to 4 hours. At specified time points (30 min,
1 hr, 4 hrs), the cells were washed with PBS and fixed with 4%
neutral buffered paraformaldehyde. To visualize anti-A33
antibodies, cells were permeabilized with a buffer of PBS
containing 0.5% BSA and 0.2% Triton-X, then stained with a 1:500
dilution of Alexa Fluor 488 labeled anti-human IgG (Invitrogen).
After staining, cells were washed and mounted in Prolong Gold
medium containing a DAPI nuclear stain (Invitrogen). SWNT-cMORF and
cMORF could be directly visualized through the appended AF647
fluorophores. Imaging was performed on a laser scanning confocal
imaging system (Leica), with settings maintained across
visualization of the different cell treatment conditions.
Cell Binding
[0065] The Daudi B-cell lymphoma, LS174T colon adenocarcinoma, and
HL60 promyelocytic leukemia cell lines were obtained from the ATCC
and cultured in RPMI containing 10% FBS at 37.degree. C. in 5%
CO.sub.2. GFP transfected Daudi were produced through retroviral
transfection as described previously (9). Prior to flow cytometric
assays cells were suspended in ice cold phosphate buffered saline
(PBS) containing 2% human serum as a blocking agent, or, where
noted, 100% human serum. Suspension cultures were washed by
centrifugation and resuspension, while adherent cells were
trypsinized in 0.05% tryspin containg EDTA (Gibco) followed by
washing. Cells in suspension were first bound with mAb-MORF at 10
nM for 1 hour, either on ice or at 37.degree. C. Following mAb-MORF
treatment, cells were washed with binding buffer, and then bound
with SWNT-cMORF-AF647 conjugates at a range of concentrations up to
10 .mu.g/mL. Cells were washed by centrifugation/resuspension and
read on an Accuri C6 flow cytometer (Accuri, Inc.). Data were
processed using Flojo analysis software (TreeStar, Inc.). A second
cell binding assay was conducted to compare cMORF-DOTA and
SWNT-cMORF-DOTA second steps. These methodologies were identical
except instead of a fluorophore, either .sup.111In or .sup.225Ac
labeled DOTA was assayed on a Cobra II gamma counter (Packard).
In Vivo Binding Studies
[0066] All mice were female NCl nu/nu, 4-6 weeks old, and all
animal studies were conducted under the approval of the
Institutional Animal Care and Use Committee. To assess binding in
the Daudi lymphoma model, Daudi cells were cultured in suspension,
followed by washing with cold PBS and resuspension in 0.9% NaCl
(Hospira, Inc.). Mice were then injected with 20 million cells per
mouse. After 6 hours, the mice were then treated with 3 .mu.g of
morpholino conjugates of either Daudi specific anti-CD20 Rituximab
(anti-CD20-MORF) or isotype control anti-CD33 HuM195
(anti-CD33-MORF). 16 hours later, mice were then injected i.p. with
2 .mu.g of SWNT-cMORF-AF647. The SWNT-cMORF-AF647 was allowed to
circulate and target for 4 hours, after which mice were sacrificed
and the lymphoma cells collected by lavage of the i.p. cavity with
ice cold PBS. This cell suspension was washed with PBS, passed
through a cell strainer to remove debris, and run on a Accuri C6
flow cytometer. The cell suspension was gated for Daudi-GFP cells
in the FL1 channel and then fluorescence of SWNT-cMORF-AF647 was
measured in the FL4 channel. Data were analyzed on FloJo cytometry
software (TreeStar, Inc.).
[0067] For solid tumor studies, 4-6 week old female NCl nu/nu mice
were xenografted with 5 million LS174T cells subcutaneously into
the right flank. Cells were suspended in a 1:1 mixture of Matrigel
matrix (BD) and ice cold PBS. Once tumors reached .about.150
mm.sup.3, at about 7 days, mice were treated with i.p. injection of
20 .mu.g of either tumor specific anti-A33-MORF or isotype control
anti-CD33-MORF antibodies diluted in normal saline. 72 hours after
treatment with the antibodies, the mice were injected with 12 .mu.g
of SWNT-cMORF-.sup.111In(DOTA) intravenously via the retroorbital
sinus. Isoflurane anesthesia was administered with a vaporizing
chamber (VetEquip). Typical specific activity of
SWNT-cMORF-.sup.111In(DOTA) was .about.2-3 Ci/g and a total
activity of 24-36 .mu.Ci per mouse was administered. At various
time points mice were sacrificed, their organs harvested and
weighed, and the radioactivity was counted on a Cobra II gamma
counter (Packard).
Biodistribution
[0068] SWNT-cMORF-DOTA were labeled with .sup.111In at 99.7%
radio-chemical purity as previously described. 30 BALB/c mice (NCl
Labs), age 4-6 weeks, were randomized into 6 groups of 5 mice each.
Each mouse was injected I.P. with 3200 nCi of
SWNT-cMORF-.sup.111In(DOTA) in 200 uL of sterile normal saline.
Mice were sacrificed at 1, 4, 8, 12, and 24 hours. Bedding from the
cages containing excreted urine and feces were collected for
quantification. Kidneys, lungs, muscle, heart, blood, liver,
spleen, intestine, and bone were harvested from sacrificed mice and
the % injected dose for each organ was quantified as previously
described.
In Vivo Toxicology Study in Mice: SWNT-cMORF-DOTA
[0069] SWNT-cMORF-DOTA were labeled with .sup.225Ac at a 98.4%
radio-chemical purity. 45 BALB/c mice (NCl Labs,) age 4-6 weeks.
Mice were randomly divided into 9 groups of 5 mice and received a
single injection as shown in Table 1.
TABLE-US-00001 TABLE 1 Cage Number I.P. injection (200 uL) 1 Saline
2 SWNT-cMORF-DOTA (0 nCi .sup.225Ac) (17 .mu.g) 3 SWNT-cMORF-DOTA
(450 nCi .sup.225Ac) (2.83 .mu.g) 4 SWNT-cMORF-DOTA (900 nCi
.sup.225Ac) (5.66 .mu.g) 5 SWNT-cMORF-DOTA (1350 nCi .sup.225Ac)
(8.5 .mu.g) 6 SWNT-cMORF-DOTA (1800 nCi .sup.225Ac) (11.3 .mu.g) 7
SWNT-cMORF-DOTA (2250 nCi .sup.225Ac) (14.2 .mu.g) 8
SWNT-cMORF-DOTA (2700 nCi .sup.225Ac) (17 .mu.g) 9 Unchelated
.sup.225Ac (1350 nCi .sup.225Ac) (3.57 .mu.g)
[0070] Bedding was collected in full from each group at 10 hours to
determine elimination. Each group was visually assessed and weighed
regularly over 140 days at which time the liver, spleen, bone
marrow, and kidneys of each mouse were submitted for histological
analysis. Hematoxylin and eosin (H&E) staining of
paraffin-embedded tissue sections was performed according to
standard protocols. Histological evaluation was performed by a
veterinary pathologist blinded to the treatment groups.
Toxicology Study in Mice: Pre-Annealed Ab-MORF-SWNT-cMORF-DOTA
(.sup.225Ac)
[0071] SWNT-cMORF-DOTA were labeled with .sup.225Ac at a 99.6%
(post dialysis workup) radio-chemical purity. Anti-CD20 antibodies
(Rituximab), were labeled with MORF at 0.9 per antibody to stem
crosslinking. SWNT-cMORF-(.sup.225Ac)DOTA were placed in a 20,000
MWCO dialysis cassette with a MORF to cMORF excess of Ab-MORF
(Rituximab). This was allowed to stir in metal free water for 6
hours, replacing water twice, before collection of the annealed
product by syringe. HPLC confirmed annealing. 35 BALB/c mice (NCl
Labs,) age 5-7 weeks. Mice were randomly divided into 7 groups of 5
mice and received a single injection as shown in Table 2. Mice were
followed until death or weight stabilization had occurred. Injected
masses denote mass of complete construct, though up to one
equivalent mass of excess, unbound Ab-MORF was also injected in
groups 3-7.
TABLE-US-00002 TABLE 2 Cage Number I.P. injection (200 uL) 1 Saline
2 Ab-MORF (50 ug) 3 Ab-MORF-SWNT-cMORF-DOTA (0 nCi .sup.225Ac) (~24
ug) 4 Ab-MORF-SWNT-cMORF-DOTA (450 nCi .sup.225Ac) (~6 ug) 5
Ab-MORF-SWNT-cMORF-DOTA (900 nCi .sup.225Ac) (~12 ug) 6
Ab-MORF-SWNT-cMORF-DOTA (1350 nCi .sup.225Ac) (~18 ug) 7
Ab-MORF-SWNT-cMORF-DOTA (1800 nCi .sup.225Ac) (~24 ug)
In Vivo Therapy Study in Mice
[0072] SWNT-cMORF-DOTA were labeled with .sup.225Ac at a 96%
radio-chemical purity. The Morpholino-tagged rituximab (3.5 MORF
per antibody) was produced as previously detailed. Binding
selectivity of the reagents was confirmed in vitro using .sup.111In
labeled SWNT-cMORF-DOTA as a the reporter in a cross control with
HL60 cells and morpholino tagged Hum195 (3.74 MORF per antibody).
CB17SC-F SCID mice (Taconic Labs,) age 5-7 weeks, were injected
I.P. with 20 million firefly-luciferase expressing Daudi lymphoma
cells in 200 .mu.L PBS. After one week, the mice were imaged to
confirm growth of tumor in all mice to be used in the therapy
experiment using an IVIS 200 Imaging System (Caliper Sciences).
Images were performed 5 min after an IP injection of 200 .mu.L of 5
mg/mL luciferin.
[0073] The mice were then randomly assigned into 10 treatment
groups of 5 mice each that received two sequential injections at 0
and 24 hours as shown in Table 3.
TABLE-US-00003 TABLE 3 Injection 1, Injection 2, Group 0 h (200 uL)
24 h (200 .mu.L) 1: Untreated Saline Saline control 2: Anti-CD20
1.5 ug anti-CD20-MORF Saline alone control 3: Unlabeled Saline
SWCNT-cMORF-DOTA SWNT Control (0 nCi .sup.225Ac) (3.57 .mu.g) 4:
Isotype 1.5 ug anti-CD33-MORF SWCNT-cMORF-DOTA control (333 nCi
.sup.225Ac) (1.19 .mu.g) 5: Isotype 1.5 ug anti-CD33-MORF
SWCNT-cMORF-DOTA control (666 nCi .sup.225Ac) (2.38 .mu.g) 6:
Isotype 1.5 ug anti-CD33-MORF SWCNT-cMORF-DOTA control (999 nCi
.sup.225Ac) (3.57 .mu.g) 7: Treatment 1.5 ug Anti-CD20-MORF
SWCNT-cMORF-DOTA group (Rituximab) (333 nCi .sup.225Ac) (1.19
.mu.g) 8: Treatment 1.5 ug Anti-CD20-MORF SWCNT-cMORF-DOTA group
(Rituximab) (666 nCi .sup.225Ac) (2.38 .mu.g) 9: Treatment 1.5 ug
Anti-CD20-MORF SWCNT-cMORF-DOTA group (Rituximab) (999 nCi
.sup.225Ac) (3.57 .mu.g) 10: Blocked 1.5 ug Anti-CD20-MORF MORF
Blocked treatment group (Rituximab) SWCNT-cMORF-DOTA Dual control
(999 nCi .sup.225Ac)
[0074] Mice were imaged and photons quantitated for tumor burden on
day 3, 6, 9 and 15. Data were analyzed using Igor Pro Living Image
software Version 2.60 (Wavemetrics.)
Example 2
Oligonucleotide Modification and Radiolabeling of SWNT
[0075] The amine functionalized single-walled carbon nanotubes
(FIG. 1A, 1) used herein were prepared from high purity
arc-produced single-walled carbon nanotubes or HiPCO SWNT via
covalent cycloaddition of azomethine ylides as described (27-28).
This reaction attaches hydrophilic chains to the single-walled
carbon nanotubes sidewalls that could be terminated with primary
amines to serve as the attachment site for bifunctional radiometal
chelates, fluorophores, and the morpholino oligonucleotide
complementary to a modified antibody (cMORF).
[0076] Constructs averaged 350 nm in length by DLS (Zetasizer Nano
Zs system equipped with a narrow bandwidth filter (Malvern
Instruments)) and TEM (EM-201 (Philips)) with diameter of
approximately 1.2 nm giving 12 carbon atoms per 2.5 angstroms. They
were characterized by Raman spectroscopy (InVia micro Raman system
(Renishaw)).
[0077] In order to append the morpholinos to both the targeting
antibodies and the carbon nanotubes, a chemical approach that
produces a spectrally quantifiable bis-aryl hydrazone linkage
between the two entities was chosen (6, 29). The side-wall amines
on the single-walled carbon nanotubes were reacted with an
activated ester of an aromatic aldehyde linker at a
sub-stoichiometric ratio to introduce a number of aldehydes onto
the SWNT-NH.sub.2 sidewalls. The product 2 (FIG. 1A) was then
analyzed for amine content by a quantitative ninhydrin assay (30)
which showed that approximately half of the amines remained
un-reacted, (1.1 reacted:1 unreacted, starting with 1.1 mmol/L
primary amines). Based on amine to carbon molar ratios (discounting
the weight of SWNT covalent adducts) the level of substitution is
approximately 1 addition per 289 carbons or approximately 125
morpholino adducts per SWNT of median length (348.9 nm). The
average unmodified and modified nanotube molecular weights
(434,968.20 g/mol, .about.1.22E6 g/mol) derivation is provided as
follows and assumes a SWNT with a diameter of 1 nm and length of
348.9 nm determined by dynamic light scattering.
[0078] For an unmodified tube: (1 nm/0.245 nm)(3.1414)(2 carbon
atoms)=25.64 carbons around the circumference. For every 0.283 nm
length, there are 4.times.25.64=102.58 carbon atoms. (100 nm/0.283
nm)(102.58)(12.01)=434,068.20 Amu.
[0079] For a modified tube bearing .about.125 morpholino adducts:
.about.6317 Amu.times.125=789625 Amu.
789625+434968=1.22.times.10.sup.6 Amu.
The incorporated aromatic aldehydes were quantified by reaction
with 2-hydrazino pyridine to form a spectrally quantifiable
chromophore of .lamda..sub.max=350 nm. This reaction confirmed that
the reacted amines had been converted to reactive aldehyde linker
groups at an approximate 50% yield.
[0080] Custom synthesized morpholinos, with sequences chosen based
on previous pretargeting literature (31), bearing 3' primary amines
(cMORF, sequence: TAG-TTG-TGA-AGT-AGA-AGA; SEQ ID NO: 2) were
reacted with a 20-fold excess of succinimidyl hyzdrazino
nicotinamide at pH 9 and purified via gel filtration chromatography
to yield the cMORF-HyNic product. The cMORF-HyNic was coupled with
the aldehyde functionalized single-walled carbon nanotubes at pH
5.5 to yield the SWNT-cMORF conjugate 3 (Fig. A). The remaining
amines in compound 3 were then either capped with the radiometal
chelating moiety,
(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic acid (DOTA), for subsequent labeling with radiometals (FIG. 1B,
5), or reacted with the activated ester of Alexa Fluor 647 to
introduce a fluorescent label for microscopy and cytometric assays
(FIG. 1B, 4). The modification of post-morpholino-addition SWNT
with DOTA or Alexa Fluor 647 was run with stoichiometric excess of
the new adduct. This reaction consumed all remaining amines to
yield 1 DOTA or Alexa Fluor per 316 carbon atoms or approximately
115 adducts per median-length tube (discounting the weight of SWNT
covalent adducts).
[0081] The DOTA chelator was labeled with isotopes befitting their
intended use. The gamma emitting isotope .sup.111In was used for
biodistribution and binding studies. Single-walled carbon nanotubes
also were labeled with .sup.225Ac, an alpha-particle emitting
cytotoxic isotope, for application in therapeutic models. The DOTA
functionalized material 5 was radiolabeled with .sup.111InCl.sub.3.
The radiolabeled product 6 was typically produced with .about.95%
radiochemical purity. Using this synthetic approach, SWNT-cMORF
conjugates of high specific activities, up to 25 Ci/g, were
produced when labeling with .sup.111In. When labeling
SWNT-cMORF-DOTA, 5, with varying amounts of .sup.225Ac, specific
activities up to 2 Ci/g were achieved. This represents a greater
than 100-fold improvement in specific activity of the SWNT-cMORF
compared to monoclonal antibodies (10, 32) of similar molecular
weight, and demonstrates the potential for signal amplification
with highly multivalent single-walled carbon nanotubes
scaffolds.
[0082] HPLC of constructs 4, 5, and 6 in both reverse phase and gel
permeation systems demonstrated high purity and the single peak
observed had a spectrum consistent with single-walled carbon
nanotubes appended with bis-aryl hydrazone linkages of
.lamda..sub.max=354 nm (FIG. 1C). The Raman spectrum of the amine
functionalized SWNTs, 1, was consistent with highly modified SWNTs,
demonstrating a rise in the disorder band at 1360 cm-1 and a broad
major tangential mode peak at 1580 cm.sup.-1, as in previous
studies (8).
[0083] Antibodies were coupled to morpholino oligonucleotides
complementary to the sequence on the nanotubes via similar reaction
chemistry to that implemented for the nanotube conjugation. Four
different antibody-MORF conjugates were produced, anti-CD33-MORF
(Lintuzumab, 3.75 MORF per Ab), anti-CD20-MORF (Rituximab, 3.5 MORF
per Ab), and anti-CD and anti-A33-MORF (huA33). All three
antibodies are of the human IgG1 isotype and are extensively used
in human therapies. This approach to attachment of morpholino
oligos to antibodies has been described (29) and typically resulted
in 3 to 6 morpholinos per antibody, regardless of the antibody
type. The product antibody-morpholino conjugates (mAb-MORF) were
>95% purity, as measured by size-exclusion HPLC. FIG. 1D is a
diagram of SWNT-cMORF self-assembly onto mAb-MORF targeted tumor
cells.
Example 3
SWNT-cMORF Hybridizes with Multiple Different mAb-MORF In Vitro and
in the Presence of Serum at 37.degree.
[0084] Hybridization of the antibody-MORF onto the SWNT-cMORF
conjugate could be monitored through HPLC (FIGS. 2A-2D). The
mAb-MORF conjugates alone had an elution time of approximately 18
minutes, which was consistent with a molecular weight of
.about.150,000 Da based on protein standards. When the mAb-MORF,
anti-CD20-MORF and anti-A33-MORF, was incubated with the
complementary SWNT-cMORF construct, the nanotube elution shifted to
a high-molecular-weight band and eluted at the void volume at 12
min. (FIG. 2B). The column's molecular weight cutoff is 600 kDa,
suggesting that multiple antibodies were hybridizing to the
single-walled carbon nanotubes to form large, but still soluble,
multimeric constructs (FIG. 2A).
[0085] In order to specifically monitor the elution of the
SWNT-cMORF through an appended label, a similar experiment was
performed with radiolabeled SWNT-cMORF-.sup.111In(DOTA) (FIG. 2B,
6), and the elution of the isotope was monitored with an HPLC
radiodetector which showed an identical pattern of results for
SWNT-cMORF mixed with both anti-CD20-33 MORF and anti-A33-MORF.
This confirmed that the assembly of these complexes is independent
of the antibody used (FIG. 2C) and the presence of 100% human serum
at 37.degree. C. (FIG. 2D). The dependence of the observed
phenomenon on MORF/cMORF hybridization was demonstrated by blocking
the nucleotide hybridization sites of the Ab-SWNT complex by
addition of excess free cMORF.
[0086] Another approach to assay the hybridization of the
SWNT-cMORF-.sup.111In(DOTA) conjugate and complementary mAb-MORF
was to capture the radiolabeled SWNT-cMORF with protein A beads
after addition of mAb-MORF (FIG. 2E). Because protein A selectively
binds IgG, the labeled SWNT-cMORF should be immobilized on the
agarose beads only when annealed to the IgG. Indeed, when
radiolabeled SWNT-cMORF conjugates were incubated with protein A
beads alone, only 1% of the nanotubes were captured by beads.
However, when complementary mAb-MORF was previously added to the
SWNT-cMORF, .about.35% of the nanotubes were captured by the beads.
This binding to the beads could be completely blocked by addition
of excess cMORF to block nanotube hybridization sites on the
antibody. This hybridization could occur and be maintained in the
presence of 100% serum at 37.degree. C. for 24 hours. Nearly
identical binding was obtained under these conditions as compared
to binding buffer (PBS with 1% BSA), demonstrating again that serum
and temperature had little to no effect on the nanotube-antibody
hybridization.
[0087] The efficiency of hybridization was unaffected by serum, and
could be blocked by an addition of excess free cMORF. Isocratic,
aqueous mobile phase elution of the SWNT-cMORF conjugate in the
HPLC size-exclusion column (Superdex 200) occurred as a late, broad
band. The delayed elution, despite the high single-walled carbon
nanotubes molecular weight, is attributable to the highly
anisotropic geometry of the SWNT, which is known to significantly
retard elongated macromolecules in common gel (9, 33). This peak
had spectral features consistent with SWNT. It was found that if
mAb-MORF (anti-CD20-MORF or anti-CD33-MORF) and SWNT-cMORF were
mixed together at micro-molar concentrations for 12 h at 4.degree.
C., they formed sub-millimeter, visible aggregates. Neither
SWNT-cMORF alone, nor mAb-MORF alone at 10.times. this
concentration, showed any aggregation. When the macrostructures
were administered to cells, these aggregates showed no targeted
binding enrichment in a cross control with HL60 or Daudi cells.
This suggested that due to the multivalent nature of the
SWNT-cMORF, extensive cross-linking had occurred, perturbing the
ability to bind to multiple cells, or resist washes before flow
cytometric analysis.
Example 4
Self-Assembly on Pretargeted Cells
[0088] SWNT-cMORF self-assembled onto tumor cell surfaces that were
bound specifically by mAb-MORF conjugates containing the
complementary oligonucleotide sequence. Three different cancer cell
lines, Daudi (B-cell lymphoma), HL60 (promyelocytic leukemia) and
LS174T (colon adenocarcinoma), pretargeted by anti-CD20
(Rituximab), anti-CD33 (Hum195), and anti-A33 monoclonal
antibodies, respectively, were used. All antibodies were human IgG1
isotype. The cells were incubated with either specific or isotype
control antibody-MORF, followed by treatment with fluorescently
labeled SWNT-cMORF (FIG. 1B, 4). As a control, cells targeted with
specific mAb-MORF conjugates were blocked with excess cMORF prior
to the addition of SWNT-cMORF. The binding of fluorescently labeled
single-walled carbon nanotubes was quantified as the change in
median fluorescence intensity as measured by flow cytometry.
[0089] The single-walled carbon nanotubes self-assembled onto all
three cell types with excellent specificity (FIGS. 3A-3D).
Furthermore, the binding was significantly abrogated when blocked
with excess cMORF and there was little binding to cells treated
with isotype control mAb-MORF. Binding also was tested across a
range of conditions, at temperatures of 4.degree. C., 25.degree.
C., and 37.degree. C. as well as in 100% human serum. Similar
binding was obtained regardless of the binding conditions,
confirming that MORF/cMORF hybridization onto cells can occur in
serum at 37.degree. C.
[0090] To test the affinity of the interaction between the
SWNT-cMORF and the cells pre-targeted with mAb-MORF, a similar
binding study was performed (FIG. 3E). After subtraction of the
non-specific binding, calculated using isotype control
anti-CD33-MORF, the shift in median fluorescence was plotted as a
function of the SWNT-cMORF concentration. An avidity estimation was
performed targeting Daudi cells with anti-CD20-MORF, HL60 cells
with anti-CD33-MORF and LS174T with anti-CD33-MORF. The result was
a characteristic sigmoid binding curve, with an apparent
dissociation constant of 0.3 .mu.g/mL. With the expected molecular
weight of the SWNT-cMORF constructs of 350-500 kDa, this apparent
affinity represents a dissociation constant of .about.0.6 nM. This
affinity was identical for the three different cell types,
demonstrating that the affinity of the SWNT-cMORF for the mAb-MORF
was independent of the chosen cancer target or targeting
vehicle.
Example 5
Self-Assembly on Cells Leads to Capping and Internalization
[0091] After demonstration of self-assembly of SWNT-cMORF onto
tumor cells, the fate of the self-assembled SWNT-(cMORF-(mAB-MORF))
complexes on the cell surface was determined. A series of confocal
microscopy studies was performed to track anti-A33 antibodies and
anti-A33/single-walled carbon nanotubes complexes after binding to
the LS174T cells. Plated cells were treated with anti-A33 or
anti-A33-MORF, washed, and incubated at 37.degree. C. in culture
medium for up to 24 hours.
[0092] When cells were treated with the anti-A33 antibody alone
(green), the antibody was stable on the cell surface for up to 24
hours (FIG. 4A). A33 was chosen for this demonstration due to
stable surface expression of A33 glycoprotein after anti-A33
antibody binding, its slow internalization and lysosomal
degradation with a turnover of 6 weeks at 37.degree. C. (34)
similar to CD20 as used in the therapy studies. The antibody was
evenly distributed along the membrane. Likewise, the anti-A33-MORF
antibody conjugate alone was stable on the cell surface and
remained evenly distributed (FIG. 4B). There was no binding of an
isotype control antibody (anti-CD19-MORF). However, when SWNT-cMORF
(pink) was added to anti-A33-MORF pre-targeted cells, followed by
up to 4 hours of incubation at 37.degree. C. a dramatic change was
observed in the distribution of antibody in the target cells (FIGS.
4C, 4E) including clustering of the self-assembled
mAb-MORF/SWNT-cMORF complexes on the cell membrane, leading to
punctate staining suggestive of antigen capping. Addition of
SWNT-cMORF to cells that were targeted with control antibodies
(anti-A33 without MORF or isotype control anti-CD19-MORF) did not
result in any change in staining pattern and no binding of
SWNT-cMORF to the control antibody treated cells was observed.
[0093] Furthermore, in cells treated with mAb-MORF followed by
SWNT-cMORF the appearance of antibody and nanotube positive,
punctate structures were observed inside the cells (FIG. 4D). This
clustering phenomenon was evident 30 minutes after addition of
SWNT-cMORF constructs, and intracellular staining continued to
increase through the 4 hours assayed. These images suggest an
endocytic uptake of the anti-A33-MORF/SWNT-cMORF complexes upon
self-assembly into multimeric surface structures separate from
internalization of individual A33 molecules. Therefore, as
SWNT-cMORF constructs can bind multiple antibodies on the cell
surface, this cross-linking apparently leads to promotion of rapid
intracellular delivery of the complexes in line with observations
of crosslinking upon prolonged mixing of SWNT-cMORF and Ab-MORF
(FIGS. 1A-1D), a desired, but unexpected effect.
[0094] To test this hypothesis, anti-A33-MORF targeted cells were
treated with free cMORF oligonucleotides, which should not bind
multiple antibodies. When cells pretreated with anti-A33-MORF were
subsequently treated with fluorescently labeled cMORF alone, the
staining profile of the cMORF was completely co-localized with the
antibody and the pattern of surface binding was unchanged from
antibody alone (FIG. 4D, compare with FIGS. 4A-4B). This result
suggests that the capping and internalization phenomenon depended
on the multivalency of the second step.
Example 6
Self-Assembly In Vivo in Mice
[0095] Having demonstrated that the SWNT-cMORF can self-assemble
onto specific cancer cells in vitro, this activity was assessed
next in live mice. This was initially performed in a CD20-positive
Daudi lymphoma model. SCID mice were injected with Daudi-GFP (9)
cells i.p, followed 24 hours later with injection of either
lymphoma specific anti-CD20-MORF or anti-CD33-MORF isotype control.
The mice were treated 24 hr later with fluorescently labeled
SWNT-cMORF. After a further 6 hours, the mice were sacrificed, the
lymphoma cells were harvested and the cell suspension was analyzed
by flow cytometry. The Daudi-GFP cells were tracked by gating for
green positive cells and then assayed for the fluorescence of the
SWNT-cMORF-Alexa Fluor 647 in the FL4 channel. The SWNT-cMORF bound
selectively to the tumor cells in mice pretargeted with specific
antibody as evidenced by the 20-fold increase in the median
fluorescence intensity of the gated Daudi cells was observed in the
FL4 channel (FIGS. 5A-5B) from the control anti-CD20 mAb-MORF
treated animal, confirming specific binding to the target in the
harvested cells. The isotype control antibody treated animals did
not demonstrate binding or self-assembly.
[0096] It also was demonstrated that this approach is feasible with
systemically administered constructs in a solid carcinoma model in
which tumor penetration of each of the components would be more
difficult. A subcutaneous, xenografted, solid tumor LS174T colon
adenocarcinoma model was chosen. The anti-A33 antibody tumor
targeting in this model was separately tested via positron emission
tomography of 1-124 labeled anti-A33. This demonstrated that, by 72
hours after injection, the antibody reaches .about.10% ID/g in the
tumor and was reduced to .about.4% ID/g in the bloodstream (tumor
to blood ratio of .about.2.5).
[0097] These relative concentrations are sufficient for
administration of the SWNT-cMORF-(.sup.111In)DOTA second step at 72
hours after mAb-MORF. Following treatment with i.v. injection of
SWNT-cMORF-(.sup.111In)DOTA, the accumulation of
SWNT-cMORF-(.sup.111In)DOTA in the blood, tumor, and muscle were
quantified at 4 and 24 hours. As in previous biodistribution
studies with functionalized single-walled carbon nanotubes
constructs, the SWNT-cMORF-(.sup.111In)DOTA rapidly cleared the
bloodstream with levels of 1.4% ID/g and 0.26% ID/g at 4 and 24
hours, respectively. There was no significant difference in blood
levels between experimental treatment and the isotype control
group.
[0098] At 24 hours, SWNT-cMORF was better retained in the tumors of
specifically targeted animals (FIG. 5C) and there was a modest, but
significant increase in the tumor to blood ratio for the
specifically targeted tumors (3.2) versus the isotype control
(1.7). The tumor to blood ratio at this point was slightly improved
as compared to what was obtained with directly labeled anti-A33
antibodies at 72 hours post-injection (3.2 vs 2.5). Unlike the
tumor in FIG. 5C, no other measured tissues showed significant
enhancement of specifically treated cells versus the isotype (FIG.
5D). The non-specific tumor accumulation in control antibody
treated animals was attributed to the enhanced permeability and
retention effect, which is known to passively accumulate carbon
nanotubes in tumors (4). A panel of normal tissues was also
harvested and demonstrated no significant difference between the
two groups in any off-target sites, confirming the
tumor-specificity of this increased accumulation. As with other
covalently functionalized single-walled carbon nanotubes constructs
(8), off-target accumulation in the kidneys, liver, and spleen also
was observed.
Example 7
SWNT-cMORF-DOTA as a Delivery Vehicle for .sup.225Ac Mitigates
Radioisotope Toxicity
[0099] Having demonstrated the ability to target both solid and
disseminated malignancies, it is demonstrated that the SWNT-cMORF
constructs are effective carriers in a therapeutic model of human
lymphoma using a xenografted SCID mouse model. A key property of
any pretargeted vehicle for radio-immunotherapy is that the rapid
clearance of the secondary agent offers improved therapeutic index
due to reduced circulation time of a cytotoxic effector. A number
of studies were performed previously demonstrating that covalently
functionalized SWNT, even after addition of 18-mer
oligonucleotides, have this rapid clearance (35). Similar
biodistribution experiments performed with the constructs described
herein demonstrated similar clearance properties. Biodistribution
of SWNT-cMORF-(.sup.111In) in vivo was assessed both as total
radiation and percent injected dose at 1, 4, 8, 12, and 24 hours
after injection. As expected, the majority of the total injected
dose (.about.77% ID) was excreted (largely in urine) by 24 hours
and no organ other than the kidney had more than 0.2% of the
injected dose per gram after 1 hour. The preponderance of remaining
radiation localized to the kidneys (2.96% ID).
[0100] Rapid clearance of radiolabeled SWNT markedly reduces
toxicity in mice when compared to similarly radiolabeled
single-step monoclonal antibodies, free .sup.225Ac or antibodies
labeled with MRF directly. For monoclonal antibodies directly
labeled with .sup.225Ac, the maximum tolerated dose in mice is
400-500 nCi, usually 450 nCi, in a single injection, with acute
toxicity and death resulting from marrow failure and longer-term
renal toxicity from radioisotope daughters (36-37), whereas for
.sup.225Ac alone (FIG. 6A) this does is rapidly lethal.
[0101] Therefore, to demonstrate reduction in toxicity as compared
to similarly labeled antibodies, the total amount of radioactivity
administered ranged from 0 to 2700 nCi of .sup.225Ac. Although
dose-dependent weight loss was observed in mice receiving the
radiolabeled SWNT-cMORF-.sup.225Ac(DOTA) (FIG. 6A), all mice
administered with SWNT-cMORF-.sup.225Ac(DOTA) below 2250 nCi
survived through the 140 day experiment. One of five mice and two
of five mice died in the 2250 nCi and 2700 nCi dose level,
respectively. Mice at dose levels of 1,350-2,700 nCi presented with
limited dose-dependent toxicity, including reduced bone marrow and
splenic cellularity and smaller glomeruli at 140 days. Mice at all
doses with the exception of the free .sup.225Ac group demonstrated
normal grooming and feeding behaviors.
[0102] The pre-annealed single-step toxicity study conducted for
comparison demonstrated that all dosing groups (450-1,800 nCi)
reached a dose-dependent, terminal morbidity starting at 6 days
post-injection, presumably resulting from the inability of the
annealed structure to clear the kidneys (FIG. 6B). The maximum
tolerated dose of the pre-annealed material (<450 nCi) was
reached at a dose at least sixfold lower than when using the
nanotube carrier (>2,700 nCi) and median survival was at least
10 times shorter (<2 weeks versus >20 weeks).
[0103] Modest dose-dependent weight loss was seen in the kidneys,
liver, and spleen (the major sites of nonspecific uptake of the
SWNT constructs) (FIG. 6C). As a comparison, 450 nCi of free
actinium proved uniformly lethal at 4 days. Unlabeled
nanotube-oligonucleotide conjugates were found to have no
observable toxicity. Mice at dose levels of 0, 450, and 900 nCi
showed no discernible organ damage upon microscopic histochemical
evaluation, whereas at levels of 1350 to 2700 nCi organs showed
limited dose-dependent toxicity including reduced bone marrow and
splenic cellularity and smaller glomeruli at 140 days.
Example 8
Therapy with SWNT-cMORF-(.sup.225Ac)DOTA
[0104] The high specific activity SWNT-cMORF-.sup.225Ac is rapidly
cleared and did not produce overt toxicity up to 1350 nCi. Mice
were implanted with Daudi lymphoma cells in the peritoneal cavity
followed by multistep therapy one week later, after tumor
confirmation. The study included 10 groups of 5 mice each,
consisting of 3 treatment groups and 7 control groups for
SWNT-cMORF-.sup.225Ac(DOTA) therapy of Daudi lymphoma as shown in
Table 3 in Example 1. Tumor imaging studies demonstrated effective
dose-dependent therapy in all 3 treatment groups (FIGS. 7A-7B).
[0105] There was complete elimination of tumor burden in treatment
groups 8 and 9 (666 nCi SWNT-cMORF-.sup.225Ac(DOTA) and 999 nCi
SWNT-cMORF-.sup.225Ac(DOTA). Mice in the saline control, and cold
SWNT control showed rapid progression of tumor burden. Mice treated
with anti-CD20-MORF alone showed a brief reduction in tumor load
that was attributed to a transient response to the unlabeled
anti-CD20 antibody, which is known to be therapeutic (Rituximab).
Mice treated with the isotype control anti-CD33-MORF followed by
SWNT-cMORF-.sup.225Ac(DOTA) at several dose levels showed transient
responses, attributable to non-specific irradiation from
.sup.225Ac. Finally, to demonstrate that the therapeutic effect was
due to self-assembly and not simply additive effects of each
component of the therapy, a dual control of anti-CD20-MORF followed
by SWNT-cMORF-.sup.225Ac(DOTA) that had been mixed with excess free
MORF to block hybridization was included. In this group, four of
five mice had marked tumor progression, while one mouse had a
therapeutic response attributed to the additive effects of ADCC and
non-specific radiation.
[0106] The following references are cited herein: [0107] 1.
Scheinberg et al. Nat Rev Clin Oncol, 7:266-276 (2010). [0108] 2.
Davis et al. Nature Reviews Drug Discovery, 7:771-782 (2008).
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et al. Nat Nano, 2:47-52 (2007). [0112] 6. Villa et al. ACS Nano,
null-null (2011). [0113] 7. Villa et al. Nano Lett, 8:4221-4228
(2008). [0114] 8. McDevitt et al. PLoS ONE, 2:e907 (2007). [0115]
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[0144] One skilled in the art will appreciate readily that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those objects,
ends and advantages inherent herein. The present examples, along
with the methods, procedures, treatments, molecules, and specific
compounds described herein are presently representative of
preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Changes therein and
other uses will occur to those skilled in the art which are
encompassed within the spirit of the invention as defined by the
scope of the claims.
Sequence CWU 1
1
2118DNAArtificial Sequencemorpholino oligonucleotide sequence MORF
1tcttctactt cacaacta 18218DNAArtificial Sequencecomplementary
morpholino oligonucleotide sequence cMORF 2tagttgtgaa gtagaaga
18
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